FXR genomics in NAFLD

Non-alcoholic fatty liver disease (NAFLD) is a worldwide growing medical and socioeconomical health problem with limited existing opportunities for pharmacological interventions. Nuclear receptors (NRs) are promising pharmaceutical targets for NAFLD. NRs bind to distinct DNA binding sites and thereby regulate gene transcription. However, recent findings indicate that NR binding sites and regulated genes are not consistently retained across species and are profoundly affected by the metabolic background such as by NAFLD. These findings present a significant obstacle to efforts to translate basic NR functional studies from animal models into effective pharmacological approaches in diseased patients. Therefore, the central goal of this project is to generate the first genome-wide DNA binding profile of FXR, the most promising pharmacological NR target for NAFLD, in human liver tissue from NAFLD patients with and without pharmacological FXR stimulation. More specifically, we will test the hepatic FXR cistrome and transcriptome in normal and morbidly obese human subjects with NAFLD enrolled in clinical trials with the FXR agonist obeticholic acid (OCA). The outcome of these genomic studies will be matched to the metabolic and histological profile of those patients. In addition, we will investigate FXR interactions with the chromatin landscape in more detail using steatotic primary human hepatocytes treated with the FXR ligand OCA and novel non-steroidal FXR ligands. With this study, we hope to identify more individualized and more targeted treatment options for NAFLD. This proposal consists of three specific aims: 1.Determine NR binding motifs in regions of open chromatin in liver tissue from normal patients and patients with NAFLD. We will isolate and analyze open chromatin using the FAIRE- Seq method. We will perform motif analysis for unknown transcription factor binding sites first, followed by a specific motif analysis using established binding motifs of the metabolic active NRs FXR, LRH-1, LXRs and PPARs. 2.Determine the specific FXR cistrome in liver tissue of normal patients and patients with NAFLD treated with and without the FXR ligand OCA. We will analyze the FXR cistrome by FXR ChIP-Seq and the transcriptional profile by RNA-Seq. The genetic profile of the cistrome and transcriptome will then be matched to the metabolic profile of those patients. 3.Determine the chromatin landscape for FXR binding and transcriptional activity under steatotic conditions. For in-depth analysis of the epigenetic requirements for FXR binding we will use human primary hepatocytes, which will be treated with fatty acids to induce macrosteatosis and different FXR agonists. Experiments will include genome wide analysis of histone modifications which positively and negatively affect NR action and analysis of polymerase II binding as marker for transcriptional activity. 1

Non-alcoholic fatty liver disease (NAFLD) is a worldwide growing medical and socio- economical health problem with limited existing opportunities for pharmacological interventions. Nuclear receptors (NRs) are promising pharmaceutical targets for NAFLD. NRs bind to distinct DNA binding sites (the cistrome) and thereby regulate gene transcription (the transcriptome). However, recent findings indicate that NR binding sites and regulated genes are not consistently retained across species and are profoundly affected by the metabolic background such as by NAFLD. These findings present a significant obstacle to efforts to translate basic NR functional studies from animal models into effective pharmacological approaches in diseased patients. Therefore, the central goal of our project was to generate the first genome-wide DNA binding profile of FXR, the most promising pharmacological NR target for NAFLD, in human liver tissue from NAFLD patients with and without pharmacological FXR stimulation. We tested the hepatic FXR cistrome and transcriptome in normal and morbidly obese human subjects with NAFLD who have been enrolled in clinical trials with the FXR agonist obeticholic acid (OCA) and matched the outcome of these genomic studies with metabolic profiles of those patients. Our studies revealed that FXR binding is strikingly different between obese patients with NAFLD and non-obese patients. FXR binds to significant more binding sites in obese patients suggesting different and/or additional signaling pathways in an altered metabolic background. We tested several molecular mechanisms, which could have been the driving force for these differences, but up to now, we currently do not understand what causes the striking differences of FXR binding. However, we were able to define the major downstream consequences of different FXR binding. By combining the FXR cistrome with the transcriptome, we were able to identify mitochondrial biogenesis and function as a main target of OCA action in obese patients. When we additionally determined metabolic changes after OCA treatment, we observed that OCA could rebalance the impaired redox state in obese patients. In line with the genome-wide and metabolic changes, we found in more detail, that FXR transcriptionally regulates PGC1alpha, the transcriptional master-regulator of mitochondrial biogenesis, selectively in obese conditions. Moreover, FXR regulates critical enzymes of the mitochondrial reactive oxygen defense, such as superoxide dismutase. Overall, the results from our studies suggest that the metabolic background dictates FXR binding and consequently FXR signaling in obese conditions. Consequently, treating obese patients with FXR agonists may have yet unexplored effects. One of these effects is rebalancing the redox state. Results have not yet been published but are in preparation for submission.